Real-time PCR using hybridization probes for the rapid and specific identification of Francisella tularensis subspecies tularensis

Tularemia is a plague-like infection caused by Francisella (F) tularensis classified as a biological warfare agent. F tularensis subsp. tularensis is the most virulent subspecies demanding rapid diagnosis. Typing systems for this fastidious bacterium to the subspecies level are laborious and time consuming. Therefore, the aim of this study was to develop a real-time PCR for the rapid and specific identification of F tularensis subsp. tularensis. The specificity of the assay was determined using a comprehensive panel of Francisella strains, clinically relevant bacteria, and DNA preparations of potential hosts. F tularensis subsp. tularensis was specifically detected but no other organisms. The range of linearity was determined to be 100fg to 10ng, the lower limit of detection was 25fg of DNA (13 genome equivalents). An internal amplification control PCR system targeting lambda phage DNA was included. Neither the internal amplification control nor host DNA influenced the cycle threshold values obtained for E tularensis subsp. tularensis. In conclusion, we have developed a highly sensitive and specific assay that can be integrated into real-time PCR-based identification procedures for biological agents. This is a major diagnostic improvement, as all other methods for the specific identification of F tularensis subsp. tularensis are more time consuming.